Longus, a type IV pilus of enterotoxigenic Escherichia coli, is involved in adherence to intestinal epithelial cells

Abstract

Enterotoxigenic Escherichia coli (ETEC) is the leading bacterial cause of diarrhea in the developing world, as well as the most common cause of traveler's diarrhea. The main hallmarks of this type of bacteria are the expression of one or more enterotoxins and fimbriae used for attachment to host intestinal cells. Longus is a pilus produced by ETEC. These bacteria grown in pleuropneumonia-like organism (PPLO) broth at 37 degrees C and in 5% CO(2) produced longus, showing that the assembly and expression of the pili depend on growth conditions and composition of the medium. To explore the role of longus in the adherence to epithelial cells, quantitative and qualitative analyses were done, and similar levels of adherence were observed, with values of 111.44 x 10(4) CFU/ml in HT-29, 101.33 x 10(4) CFU/ml in Caco-2, and 107.11 x 10(4) CFU/ml in T84 cells. In addition, the E9034A Delta lngA strain showed a significant reduction in longus adherence of 32% in HT-29, 22.28% in Caco-2, and 21.68% in T84 cells compared to the wild-type strain. In experiments performed with nonintestinal cells (HeLa and HEp-2 cells), significant differences were not observed in adherence between E9034A and derivative strains. Interestingly, the E9034A and E9034A Delta lngA(pLngA) strains were 30 to 35% more adherent in intestinal cells than in nonintestinal cells. Twitching motility experiments were performed, showing that ETEC strains E9034A and E9034A Delta lngA(pLngA) had the capacity to form spreading zones while ETEC E9034A Delta lngA does not. In addition, our data suggest that longus from ETEC participates in the colonization of human colonic cells.

FIG. 1.

Percentages of longus production in ETEC E9034A strains grown in different media at 37°C in 5% CO₂, showing the highest production in PPLO broth and TSAB and no production in CFA agar or in Minca minimal medium.

FIG. 2.

Percentages of longus production in ETEC E9034A and derivative strains. (A) ETEC E9034A and derivative strains were cultured in PPLO broth and CFA agar, and expression was measured by flow cytometry. No production of longus was observed in the lngA mutant. Longus production was restored in the ETEC E9034AΔlngA strain complemented with the pLngA plasmid. (B) Western blot of whole bacterial cell extracts analyzed in 16% SDS-PAGE gels using antilongus polyclonal antibodies. The ETEC wild-type strain and the lngA mutant complemented with pLngA showed the presence of a protein of 22 kDa that corresponds to the LngA pilin. The LngA pilin was not produced in the lngA mutant. Anti-DnaK antibody was used to detect the DnaK protein to ensure that equal amounts of antigen were tested.

FIG. 3.

Immunogold labeling of longus produced by ETEC strains with antilongus antibody. (A) E9034A. (B) E9034AΔlngA. (C) E9034AΔlngA(pLngA). No gold is seen associated with the bacterial cell surface recognizing the antilongus antibody specific to the fimbriae. The electron micrographs were taken at a magnification of ×19,000. The insets in panels A and B show the whole bacterium producing longus covered with gold particles.

FIG. 4.

Qualitative comparison of adherence between ETEC E9034A and derivative mutant strains to epithelial cells. (A) Adherence assays performed with colonic human cells. In the three cellular lines HT-29, T84, and Caco-2 a significant reduction in adherence is observed in the mutant strain (E9034AΔlngA). (B) Adherence assays with nonintestinal cells where no reduction of adherence is observed. The monolayers of epithelial cells were obtained at 70 to 80% confluence and were incubated with ETEC strains for 6 h at 37°C in 5% CO₂. The experiments were performed in triplicate on three different days. *, statistically significant (P < 0.0001) with respect to the values obtained from the wild type, the lngA mutant, and the lngA mutant complemented with the same gene.

FIG. 5.

Qualitative analysis and immunofluorescence of wild-type ETEC E9034A and derivative mutants. (A) Images by light microscopy show differences in adherence between ETEC and derivative strains in contact with human colonic cells. The images were taken at a magnification of ×200 in a light microscope. (B) Images by IFM of ETEC E9034A and E9034AΔlngA(pLngA) strains showing long fluorescent fibers (green) extending several micrometers throughout the cell monolayer interconnecting the bacteria (red). No fluorescent fibers are seen in the E9034AΔlngA strain, confirming the lack of the lngA gene. DNA in bacteria and eukaryotic cells were stained red with propidium iodide. The images were processed at ×200. In both panels HT-29 cells without infection were used as controls.

FIG. 6.

Immunofluorescence and quantification of bacterial adherence to HT-29 cells by ELISA. The strains were incubated with human colonic HT-29 cells for 6 h and were read by an ELISA reader. (A) After this incubation time, the nonbinding bacteria were removed, and the cells with attached bacteria were washed with PBS. The presence of the bacteria attached to the cells and the pili were read at 405 nm using antilongus and anti-ETEC antibodies, respectively. A significant reduction in the E9034ΔlngA was also observed, as described in the legend of Fig. 4. (B) We observed that not all the bacteria [E9034A, E9034AΔlngA(pLngA)] in the supernatants produced longus. Interestingly, E9034AΔlngA showed high levels of bacteria in the supernatants in comparison to E9034A and E9034AΔlngA(pLngA) strains. *, statistically significant (P < 0.0001) with respect to the values obtained without the presence of antibodies against longus.

FIG. 7.

Inhibition assays with antilongus antibodies. (A) HT-29 cells were used to perform this assay at 1:10, 1:50, and 1:100 dilutions. A dose-dependent inhibition can be seen in E9034A and E9034AΔlngA(pLngA) strains. Due to the inability of the E9034AΔlngA strain to assemble longus, no change was observed. (B) The bacteria were added to HT-29 cells in the absence (0) or the presence of three dilutions of the preimmune serum (1:10, 1:50, and 1:10). Error bars represent standard deviations.

FIG. 8.

Twitching motility for ETEC E9034A and derivative strains. E9034A and E9034AΔlngA(pLngA) strains display spreading zones, showing the retraction and extension of these pili. This phenomenon is not observed in the ETEC E9034AΔlngA strain. HCP from enterohemorrhagic E. coli and the hcpA mutant were used as positive and negative controls, respectively.